calcimycin and Carcinoma--Ehrlich-Tumor

The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.

Topics: Animals; Annexin A2; Annexin A3; Annexin A5; Annexins; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cattle; Chloride Channels; Female; In Vitro Techniques; Ionophores; Lung; Membrane Potentials; Mice; Oocytes; Species Specificity; Swine; Xenopus laevis

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane-fractions. Agents that increase intracellular cAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system by cGMP. In fact, permeant analogs of cGMP, dibutyryl cGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition of cGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca2+ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca2+ ions.

Topics: Animals; Bucladesine; Calcimycin; Calcium; Calcium-Transporting ATPases; Carcinoma, Ehrlich Tumor; Cell Membrane; Cyclic GMP; Dibutyryl Cyclic GMP; Enzyme Inhibitors; Female; Kinetics; Mice; NADH, NADPH Oxidoreductases; Neomycin; Oxidation-Reduction; Second Messenger Systems; Signal Transduction; Sphingosine; Thapsigargin

Magnesium efflux from intact Ehrlich ascites tumor cells (EATC) has been characterized under 0-trans conditions. It is shown that a magnesium-extruding mechanism operates in these cells which brings about magnesium efflux up to 20% of the cell total content. EATC magnesium efflux is independent from extracellular Ca2+ and its apparent velocity is not affected by [Mg2+]o up to 160 microM. This extrusion, however, is strictly dependent on extracellular Na+ and it is inhibited by ouabain (1 mM), amiloride (1 mM), imipramine (0.5 mM) and quinidine (1 mM). It is not affected by HMA (5-N,N-hexamethylene) amiloride) (0.5 microM) and vanadate (0.1 mM). Bumetanide (0.5 mM) enhances magnesium extrusion in these cells. EATC magnesium extrusion can be significantly stimulated by db cAMP, forskolin, and IBX (3-isobutyl-1-methyl-xanthine). The intracellular magnesium distribution, studied also by means of the ionophore A23187, is regulated by energy metabolism and cell ATP content. Altogether our data demonstrate that EATC exhibit a magnesium extrusion sustained by Na+ gradient across the plasma membrane and carried out by a Na+/Mg2+ antiport. In these cells magnesium homoeostasis results, regulated efficiently by cell ATP and cAMP content.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Triphosphate; Amiloride; Animals; Antimycin A; Bucladesine; Bumetanide; Calcimycin; Carcinoma, Ehrlich Tumor; Colforsin; Cyclic AMP; Energy Metabolism; Glucose; Homeostasis; Imipramine; Kinetics; Magnesium; Mice; Ouabain; Quinidine; Rotenone; Time Factors; Tumor Cells, Cultured

Using Ehrlich ascites tumour cells, the short-term effects of the therapeutic glucocorticoid Methylprednisolone (MP) on the cellular energy metabolism were studied. ATP-consuming processes involved in the rapid MP effects were identified indirectly from the effects of MP on cellular oxygen consumption related to the inhibition of respiration by selective inhibitors of Ca(2+)-ATPase and protein synthesis. The effects of MP on plasma membrane permeability for Ca2+ ions and phospholipid turnover were studied directly by using confocal laser scanning microscopy and tracerkinetic measurements, respectively. MP inhibited cellular oxygen consumption, suppressed the inhibitory effect of lanthanum but not that of cycloheximide on oxygen consumption, blocked the [Ca2+]i rise in response to calcium ionophore A 23187, and decreased phospholipid turnover. MP acted instantly in a dose-dependent manner. The observed effects of MP are discussed in relation to the hypothesis that the drug has direct membrane effect affecting plasma membrane permeability and function.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Carcinoma, Ehrlich Tumor; Energy Metabolism; Female; Methylprednisolone; Mice; Mice, Inbred ICR; Oxygen Consumption; Tumor Cells, Cultured

Uncouplers CCCP (2-4 microM) or DNP (200-400 microM) when added to EL-4 thymoma or Ehrlich carcinoma ascites cells initially stimulated endogenous respiration about 2-fold but then inhibited it to a first-order rate 20-25% of controls. This inhibition was accelerated by intracellular acidification or by A23187, a Ca2+/H(+)-antiporter (i.e. when mitochondrial Ca2+ efflux was stimulated) whereas Ruthenium red, an inhibitor of uniporter-driven Ca2+ efflux, significantly slowed down the effect of uncouplers. The respiratory inhibition was associated with NAD(P)H oxidation and was partially reversed by exogenous substrates (glutamine or glucose). In the permeabilized cells, endogenous and glutamine-supported respiration was inhibited by EGTA, while succinate-supported respiration was Ca2+ independent. It is suggested that mitochondrial Ca2+ is necessary for NADH-dependent respiration of tumor cells, and uncouplers inhibit it by activation of mitochondrial Ca2+ efflux.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Animals; Calcimycin; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinoma, Ehrlich Tumor; Dinitrophenols; Egtazic Acid; Hydrogen-Ion Concentration; Mitochondria; NAD; NADP; Oxygen Consumption; Ruthenium Red; Thymoma; Tumor Cells, Cultured; Uncoupling Agents

Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca2+ or Ca(2+)-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca2+ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K+ ionophore valinomycin.

Topics: Actin Cytoskeleton; Animals; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cytochalasin B; Cytoskeleton; Female; Hypotonic Solutions; In Vitro Techniques; Mice; Osmotic Pressure

It has been shown that no relation exists between [Ca2+]i and hyperthermic cell killing, although heat-induced increase of [Ca2+]i can be observed in some cell lines. When ionophores are used, dose-dependent rises in [Ca2+]i may be found. Beyond a certain threshold of ionophore-induced increases in [Ca2+]i, cells may be killed. Different threshold levels of [Ca2+]i exist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca2+]i and heat toxicity, this synergism can be explained as heat enhanced Ca2+ toxicity. In the current report, it is shown that both ionophore-induced Ca2+ toxicity (37 degrees C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca2+]i. With ionomycin concentrations up to 15 microM, no increase in [Ca2+]i was seen in cells maintained in medium without Ca2+. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing.

Topics: Animals; Calcimycin; Calcium; Calcium Chloride; Carcinoma, Ehrlich Tumor; Cell Survival; Egtazic Acid; HeLa Cells; Hot Temperature; Humans; Ionomycin; Tumor Cells, Cultured

Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba2+ and hence presumably to Ca2+. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca2+ permeability, as part of the volume-activated signal system. In isolated inside-out patches a Ca(2+)-dependent, inwardly rectifying K+ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mM K+ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K+ channel takes place after an increase in Ca2+ from 10(-7) to 10(-6) M which is in the physiological range. Patch-clamp studies in cell-attached mode show K+ channels with spontaneous activity and with characteristics similar to those of the K+ channel seen in excised patches. The single-channel conductance for outward current at 5 mM external K+ is estimated at about 7 pS. A K+ channel with similar properties can be activated in the cell-attached mode by addition of Ca2+ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K+ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K+ channels following addition of Ca2+ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K+ channels in the presence of 3 mM Ca2+ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca2+ and could not be activated by membrane stretch (suction). In cell-attached mode single-channel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca2+

Topics: Animals; Barium; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane; Chlorides; Electric Conductivity; Hypotonic Solutions; Ion Channel Gating; Ion Channels; Membrane Potentials; Mice; Potassium Channels; Tumor Cells, Cultured; Water-Electrolyte Balance

The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and

Topics: Animals; Calcimycin; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Electric Conductivity; Fluorescent Dyes; Gramicidin; Ion Channels; Membrane Potentials; Mice; Quinine; Thiocyanates; Valinomycin

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.

Topics: Animals; Bacterial Toxins; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cations, Divalent; Cell Membrane; Cell Membrane Permeability; Erythrocyte Membrane; Hemolysin Proteins; Humans; Kinetics; Melitten; Mice; Parainfluenza Virus 1, Human; Staphylococcus aureus; Zinc

The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl- transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl- transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl- conductance is increased 14- and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl- conductance is rate limiting. An A23187-induced activation of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: inhibited by quinine which blocks the Ca2+-activated K+ channel, unaffected by substitution of NO-3 or SCN- for Cl-, and inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl- conductance. The Cl- conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl- transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl- transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl- transport pathway. The activated anion transport pathway is unselective, carrying both Cl-, Br-, NO-3, and SCN-. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl- transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN-) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from 42K flux measurements at high external K+, and from net K+ flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Topics: Animals; Biological Transport, Active; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Egtazic Acid; Female; Gramicidin; Ion Channels; Kinetics; Mice; Mice, Inbred Strains; Pimozide; Potassium; Quinine; Thermodynamics; Valinomycin

N-ethylmaleimide (NEM) treatment of steady-state Ehrlich cells induces a substantial net loss of cellular KCl and cell shrinkage. The majority of the initial K loss is Cl dependent. From estimates of membrane potential it is concluded that the NEM-induced KCl loss is electroneutral. The effect of NEM on H extrusion by cells in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-containing medium showed that only an insignificant part of the K loss could be attributed to an activation of a K-H exchange system. Consequently, NEM appears to activate a K-Cl cotransport, which causes cell shrinkage. The anion preference of the K loss is Cl greater than Br much greater than SCN = NO3. NEM also seems to inhibit a Cl-dependent Na uptake previously described in shrunken cells. Addition of NEM to cells undergoing regulatory volume decrease after swelling in hyposmotic media results in a Cl-dependent acceleration of cell shrinkage, suggesting that a Cl-dependent component of K efflux is induced by NEM also in swollen cells. A Cl-dependent K efflux is also activated in Ca-depleted cells or at reduced extracellular pH after cell swelling. Under isotonic conditions activation of Cl-dependent K flux after Ca depletion or pH reduction could not be demonstrated. The combined results show that Ehrlich cells possess a latent K-Cl cotransport that becomes active after changes in the state of SH groups, regardless of the initial cell volume. A similar K-Cl cotransport is activated in hypotonically swollen cells after Ca depletion or after reduction of the extracellular pH.

Topics: Animals; Biological Transport; Biological Transport, Active; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Chlorides; Ethylmaleimide; Hydrogen-Ion Concentration; Membrane Potentials; Mice; Osmolar Concentration; Potassium; Potassium Chloride

Ruthenium red-insensitive, uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria is much slower than from rat liver mitochondria under comparable conditions. In the presence of Pi and at moderate or high Ca2+ loads, ruthenium red-insensitive Ca2+ efflux elicited with uncoupler is approximately 20 times more rapid for rat liver than Ehrlich cell mitochondria. This is attributed to resistance of tumor mitochondria to damage by Ca2+ due to a high level of endogenous Mg2+ that also attenuates Ca2+ efflux. Calcium release from rat liver and tumor mitochondria is inhibited by exogenous Mg2+. This applies to ruthenium red-insensitive spontaneous Ca2+ efflux associated with Ca2+ uptake and uncoupling, and (b) ruthenium red-insensitive Ca2+ release stimulated by uncoupling agent. The endogenous Mg2+ level of Ehrlich tumor mitochondria is approximately three times that of rat liver mitochondria. Endogenous Ca2+ is also much greater (six fold) in Ehrlich tumor mitochondria compared to rat liver. Despite the quantitative difference in endogenous Mg2+, the properties of internal Mg2+ are much the same for rat liver and Ehrlich cell mitochondria. Ehrlich ascites tumor mitochondria exhibit slow, metabolically dependent Mg2+ release and rapid limited release of Mg2+ during Ca2+ uptake. Both have been observed with rat liver and other types of mitochondria. The proportions of apparently "bound" and "free" Mg2+ (inferred from release by the ionophore, A23187) do not differ significantly between tumor and liver mitochondria. Thus, the endogenous Mg2+ of tumor mitochondria has no unusual features but is simply elevated substantially. Ruthenium red-insensitive Ca2+ efflux, when expressed as a function of the intramitochondrial Ca2+/Mg2+ ratio, is quite similar for tumor and rat liver. It is proposed, therefore, that endogenous Mg2+ is a major regulatory factor responsible for differences in the sensitivity to damage by Ca2+ and Ca2+ release by Ehrlich ascites tumor mitochondria compared to mitochondria from normal tissues.

Topics: Animals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carcinoma, Ehrlich Tumor; Magnesium; Mitochondria; Mitochondria, Liver; Nitriles; Oxygen Consumption; Phosphates; Rats; Ruthenium Red

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Bumetanide; Calcimycin; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Extracellular Space; Furosemide; In Vitro Techniques; Ion Channels; Isotonic Solutions; Mice; Osmotic Fragility; Potassium; Sodium

The intracellularly trappable fluorescent Ca2+ indicator quin-2 was used to measure free cytosolic Ca2+, [Ca2+]i, in the two highly dedifferentiated tumor cell lines, Ehrlich and Yoshida ascites carcinomas. It was found that these carcinoma cells can trap quin-2 similarly to normal cells, but [Ca2+]i was apparently significantly lower than in any normal cell tested previously with this method. By using a new lipid-soluble heavy metal chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), which crosses artificial and natural membranes, it was found that endogenous heavy metals are responsible for partially quenching quin-2 fluorescence trapped inside the cells. Although the quenching of intracellular quin-2 fluorescence is quantitatively more relevant in these ascites carcinomas, TPEN was effective also in normal cells like lymphocytes and granulocytes. Both in the normal and especially in the malignant cell lines [Ca2+]i can be grossly underestimated at low intracellular quin-2 concentrations. Endogenous heavy metal quenching is thus a potential source of artifact when [Ca2+]i is measured with quin-2. When corrected for quin-2 fluorescence quenching by intracellular heavy metals, [Ca2+]i and basic regulatory mechanisms of [Ca2+]i homeostasis in Ehrlich and Yoshida carcinomas are similar to those of nontransformed cells.

Topics: Aminoquinolines; Animals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carcinoma, Ehrlich Tumor; Cell Line; Chelating Agents; Cytosol; Digitonin; Ethers; Ethylenediamines; Homeostasis; Humans; Ionomycin; Liposomes; Mice; Sarcoma, Yoshida

Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated indicator solution in physiological saline. Using either method, arsenazo III or antipyrylazo III was internalized into Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of antipyrylazo III, the amount of indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical ATP/ADP ratios, as measured by high performance liquid chromatography (HPLC) in cell extracts. Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.

Topics: Adenine Nucleotides; Animals; Arsenazo III; Azo Compounds; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Count; Cell Membrane Permeability; Cells, Cultured; Ethers; Indicators and Reagents; Ionomycin; Kinetics; Liver; Mice; Naphthalenesulfonates; Osmotic Pressure; Spectrophotometry, Atomic

Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers , but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl-, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca2+- activated K+ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca2+ ionophore A23187 in the presence of external Ca2+, and also by a sudden increase in external Ca2+. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca2+ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl- permeability of the cell membrane there was no change in cell volume or Cl- content when the K+ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca2+ ionophore A23187 in isotonic medium promoted Cl- loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl- permeability was increased in parallel with and even more than the K+ permeability. It is proposed that separate conductive K+ and Cl- channels are activated during regulatory volume decrease by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin.

Topics: Animals; Calcimycin; Calcium; Calmodulin; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Hydrogen-Ion Concentration; Mice; Potassium; Valinomycin; Water-Electrolyte Balance

Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca2+ ionophore, A23187, plus Ca2+ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO3(-) does not substitute for Cl-; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl(-) - Cl(-) exchange, a net loss of K+ and a net gain of Na+ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca2+-activated, quinine-sensitive K+ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K+ through the Ca2+ stimulated pathway appears to be limited by Cl- conductive movements; for when NO3(-), an anion demonstrated here to have a higher conductive movement than Cl-, is substituted for Cl-, the cells will shrink when the Ca2+-stimulated K+ pathway is activated.

Topics: Animals; Calcimycin; Carcinoma, Ehrlich Tumor; Cell Membrane; Culture Media; Deoxy Sugars; Deoxyglucose; Intracellular Fluid; Kinetics; Membrane Potentials; Mice; Osmolar Concentration; Potassium Chloride; Propranolol; Quinine

The role of calcium in cell injury has been the subject of much recent investigation. The movement and redistribution of this cation from extra to intracellular compartments and the calcium shifts between intracellular compartments may well play a determinate role in the cell's reaction to injury. Therefore, data of such shifts and their correlation with morphological, biochemical and cytoskeletal studies will provide a better understanding of these processes. To study the effects of calcium regulation on acute lethal anoxic injury and the effects of inhibition of respiration with cyanide, three experimental systems were utilized: Ehrlich ascites tumor cells, isolated rabbit proximal tubule segments and suspended or cultured rat proximal tubule cells. Although our data showed no correlation between total cell calcium and cell death except in highly selected cell systems, they did indicate that calcium can be an important control variable. Therefore, massive increases in total cell calcium, as seen in Ca3(PO4)2 precipitation in mitochondria, must be a secondary event and represent the modern day equivalent of the classical dystrophic calcification seen by pathologists in the past. Although the involvement of extracellular calcium in cell death may well be significant in some cell types, redistribution of calcium within the intracellular compartments may play an even more important role.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Survival; Hydrogen-Ion Concentration; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Microscopy, Electron, Scanning; Potassium; Potassium Cyanide; Rats

Treatment of Ehrlich ascites tumor cells with the cation ionophore A23187 results in cellular accumulation of calcium from the extracellular medium only when cellular metabolism is inhibited by uncouplers and iodoacetate. In the metabolically active cells A23187 induces a substantial loss of intracellular magnesium at extracellular Mg2+ concentrations below 1 mM. By contrast, in the inhibited cells A23187 induces a conspicuous loss of magnesium at concentrations of magnesium in the medium ranging from 0.2 to 5 mM. This cellular magnesium loss is accompanied by an increase in intracellular calcium. At 10 mM external Mg2+, A23187 provokes an intracellular magnesium increase in both metabolically active and inhibited cells with a corresponding H+ ejection. Intracellular free Mg2+ in both control and metabolically inhibited cells was estimated through a null-point titration by monitoring changes in extracellular Mg2+ concentration due to the addition of A23187 to cell suspensions with the metallochromic indicator Antipyrylazo III. Free intracellular Mg2+ was 0.4 and 1.2 mM in metabolically active cells and in cells treated with metabolic inhibitors, respectively. Under these conditions the cellular ATP was 30.9 and 2.3 nmol/mg dry wt, respectively. The results lead to the conclusion that in intact Ehrlich ascites tumor cells the level of ATP mainly regulates the intracellular free Mg2+ concentration and, in turn, intracellular free Mg2+ determines the entry of calcium into the cell after A23187 addition.

Topics: Adenine Nucleotides; Animals; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chemical Phenomena; Chemistry; Homeostasis; In Vitro Techniques; Magnesium; Mice

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.

Topics: Animals; Biological Transport, Active; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Chlorides; Electron Transport; Ion Channels; Kinetics; Mice; Potassium; Propranolol; Sodium

Passive Ca2+ entry into Ehrlich ascites tumour cells has been investigated. Passive equilibrium of Ca2+ takes place in ascites tumour cells only under conditions of exhaustive energy depletion. The specific Ca2+ ionophore A23187 does not affect Ca2+ entry into ascites tumour cells under active metabolic conditions, but it increases the rate of Ca2+ equilibration in ascites tumour cells in the early stages of energy depletion. The results of the present experiments lead to the conclusion that in ascites tumour cell plasma membrane Ca2+ permeability is not a limiting step in the regulation of intracellular calcium content, while the energy-dependent Ca2+ extrusion is the main mechanism that prevents uncontrolled intracellular Ca2+ increase. The results taken together support the hypothesis that increased Ca2+ influx into the cell, caused by plasma membrane alteration, is responsible for permanently elevated mitotic activity and for deranged metabolic behavior of these neoplastic cells.

Topics: Anaerobiosis; Animals; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Kinetics; Lactates; Lactic Acid; Mice; Oxygen Consumption; Rotenone

The metabolism of calcium has been investigated in the Ehrlich Ascites Tumour Cells (ATC). ATC extrude Ca2+ actively by an energy-dependent mechanism, supported by both respiration and glycolysis. Extrusion takes place even against a very steep concentration gradient (10 mM Ca2+). Cell calcium content is decreased by monovalent cations (Na+,K+ and Li+), which act independently from their metabolic effects. La3+ inhibits ATC Ca2+ extrusion whereas Ruthenium Red slightly decreases cell calcium content. The antibiotic ionophore A 23187 strongly increases ATC Ca2+ level. the metabolism of other divalent cations (Mg2+, Sr2+ and Mn2+) has been studied. Mg2+ does not show appreciable changes in the various metabolic conditions tested, while Mn2+ and Sr2+ behave quite differently from Ca2+, suggesting a different distribution of these cations in ATC. The experimental findings indicate that Ehrlich Ascites Tumour Cells regulate their calcium content by mechanisms related to plasma membranes while the size and activity of mitochondrial compartment is of minor importance.

Topics: Animals; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cations, Divalent; Cell Membrane; Lanthanum; Magnesium; Manganese; Mice; Potassium; Sodium; Strontium

The Ca2+ ionophore A23187 increases intracellular calcium content in normal thymic cells, while it is without effect on the corresponding neoplastic cell (Ascites thymoma) and on Ehrlich ascites tumour cells. The A23187-induced total cell calcium increase in normal thymocytes takes place both in control and energy-depleted cells, while it is lacking in neoplastic cells. In addition the ionophore stimulates aerobic glycolysis of normal thymocytes, whereas it is ineffective on neoplastic cells. The study of intracellular calcium exchange properties reveals that in normal cells the ionophore A23187 provokes a 60% increase of the exchangeable pool together with a more significant, 4-fold enlargement of the unexchangeable pool. These effects are lacking in cancer cells. The data give rise to interesting considerations concerning the regulation and compartmentalization of calcium in neoplastic cells. The results will be also discussed in relation to the models that predict altered cell calcium metabolism as a cause of cancer cell high aerobic glycolysis and uncontrolled growth.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Cell Transformation, Neoplastic; Mice; Neoplasms, Experimental; Rats; Thymoma; Thymus Gland; Thymus Neoplasms

Topics: Aerobiosis; Animals; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Edetic Acid; Egtazic Acid; Glycolysis; Kinetics; Lactates; Magnesium; Mice